The predominant milk proteins in mammals are the caseins, secretory phosphoproteins synthesized during lactation. The caseins are the main source of amino acids for the nursing infant. In addition, the caseins transport the amount of calcium phosphate needed in milk for infant bone formation. The caseins, by forming micelles which entrap calcium phosphate, raise the calcium and phosphate concentrations in milk to levels well in excess of the solubility product of calcium phosphate. Among the caseins, .kappa.-casein is required for stabilizing the casein micellar structures against precipitation. Besides stabilizing micelies, .kappa.-casein has a labile peptide bond in its primary structure susceptible to cleavage by low amounts of the protease rennin which results in milk clotting. Such milk clotting increases the retention time of milk in the intestines which is believed to be of nutritional value in young animals.
Many human infants unable to be breast-fed by their mothers experience severe digestive disturbances when fed milk from dairy animals or various alternative non-milk formulas. In fact, the onset of juvenile diabetes in infants genetically predisposed to the disease may be caused by their immune response to proteins found in cow's milk (Karajalainen et al., Mew Eng. J. Med. 327: 302-307 (1992)). Only through recombinant DNA techniques can large quantities of human milk protein be made available for those infants susceptible to severe digestive disturbances and possibly juvenile diabetes.
Therefore, a need arises to isolate DNA sequences of human milk proteins for obtaining such proteins in large quantities. It is especially desirable to have available for infants large quantities of the human milk protein, .kappa.-casein. At the present time only minute amounts of human .kappa.-casein have been isolated by chromatographic means (Brignon et al., FEBS LETTERS 188: 48-54 (1985)).
One way to isolate a DNA sequence encoding a desired human milk protein such as .kappa.-casein is via cDNA cloning. In this process, messenger RNA (mRNA) is isolated from cells known or suspected of producing the desired protein. Through a series of enzymatic reactions, the mRNA population of the cells is copied into a complementary DNA (cDNA). The resulting cDNA is then inserted into cloning vehicles and subsequently used to transform a suitable prokaryotic or eukaryotic host. The resultant gene library is comprised of a population of transformed host cells, each of which contain a single gene or gene fragment. The entire library, therefore, provides a representative sample of the coding information present in the mRNA mixture used as a starting material.
Gene libraries are screened using specific nucleic acid or antibody probes. Nucleic acid probes are useful for locating cDNAs by hybridization and autoradiography techniques. This approach, however, requires previous knowledge of at least a portion of the protein's amino acid or DNA-encoding sequence. Alternatively, methods have been developed to identify specific clones by probing recombinant gene libraries with antibodies specific for the encoded protein of interest. This method can be used with "expression vector" cloning vehicles since elaboration of the product protein is required. An example of this is the bacteriophage .lambda.-gt11 system described by Young and Davis, Proc. Natl. Acad. Sci. 80:1194-1198 (1983) .
Once the cDNA is isolated, the entire cDNA sequence information can be used to identify a full length gene for insertion into transgenic animals. This leads to the production of mature proteins.